For over a dozen years, studies from Dr. Hannun's laboratory have focused on elucidating mechanisms and functions for sphingolipids in signal transduction and cell regulation. The major line of investigation has resulted in the elucidation of the sphingomyelin cycle/pathway whereby the action of several extracellular stimuli or stresses (such as TNFalpha or heat) results in activation of a neutral sphingomyelinase. This enzyme cleaves membrane sphingomyelin and causes the release and accumulation of ceramide, a candidate intracellular mediator. Multiple lines of evidence have implicated ceramide in the regulation of the stress response and in regulating cell cycle arrest, cell senescence, and apoptosis. Whereas information on the functions of ceramide and its mechanisms of action continue to accumulate, little progress has been developed concerning the regulation of neutral sphingomyelinase (N-SMase); possibly a result of the great difficulty in working with this enzyme and the lack of purified preparations. The investigator's laboratory has made important strides in the last 2-3 years in purifying the enzyme and defining several mechanisms of in vitro and cellular regulation involving glutathione (GSH) and cysteine proteases. The results have generated the following hypothesis: N-SMase is a key enzyme involved in regulating ceramide formation and in initiating ceramide-dependent pathways of cell regulation. The enzyme is regulated reversibly by GSH and irreversibly by caspases. Therefore, the specific aims are: (1) Purify, characterize, and clone neutral sphingomyelinase. (2) Determine the in vitro mechanisms of regulation of neutral sphingomyelinase by GSH, and define the mechanism and significance of this mode of regulation in cellular functions. (3) Define the in vitro and cellular regulation of neutral sphingomyelinase by the cysteine protease, caspase 7. These studies should provide a biochemical and molecular understanding of a key enzyme involved in regulated metabolism of sphingomyelin and the generation of ceramide. These studies are essential to establish and consolidate the sphingomyelin/ceramide pathway as a major mechanism in cell growth regulation. N-SMase may emerge as a key target in the understanding of the mechanisms of the stress response and in therapeutic development.